Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

BACKGROUND: Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL). METHODOLOGY/PRINCIPAL FINDINGS: We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/-0.3 n = 3), CHA6.8 (FA ratio 1.08+/-0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/-0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/-0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/-0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/-0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure. CONCLUSION: Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model

Quorum Sensing Regulation of the Two hcp Alleles in Vibrio cholerae O1 Strains

BACKGROUND: The type VI secretion system (T6SS) has emerged as a protein secretion system important to several gram-negative bacterial species. One of the common components of the system is Hcp, initially described as a hemolysin co-regulated protein in a serotype O17 strain of Vibrio cholerae. Homologs to V. cholerae hcp genes have been found in all characterized type VI secretion systems and they are present also in the serotype O1 strains of V. cholerae that are the cause of cholera diseases but seemed to have non-functional T6SS. METHODOLOGY/PRINCIPAL FINDINGS: The serotype O1 V. cholerae strain A1552 was shown to express detectable levels of Hcp as determined by immunoblot analyses using polyclonal anti-Hcp antiserum. We found that the expression of Hcp was growth phase dependent. The levels of Hcp in quorum sensing deficient mutants of V. cholerae were compared with the levels in wild type V. cholerae O1 strain A1552. The expression of Hcp was positively and negatively regulated by the quorum sensing regulators HapR and LuxO, respectively. In addition, we observed that expression of Hcp was dependent on the cAMP-CRP global transcriptional regulatory complex and required the RpoN sigma factor. CONCLUSION/SIGNIFICANCE: Our results show that serotype O1 strains of V. cholerae do express Hcp which is regarded as one of the important T6SS components and is one of the secreted substrates in non-O1 non-O139 V. cholerae isolates. We found that expression of Hcp was strictly regulated by the quorum sensing system in the V. cholerae O1 strain. In addition, the expression of Hcp required the alternative sigma factor RpoN and the cAMP-CRP global regulatory complex. Interestingly, the environmental isolates of V. cholerae O1 strains that showed higher levels of the HapR quorum sensing regulator in comparison with our laboratory standard serotype O1 strain A1552 where also expressing higher levels of Hcp

Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC) from V. cholerae strains

Background Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied. Methodology/Principal Findings OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor. Conclusion/Significance Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy

Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae

Background Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. Methodology/Principal Findings In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37. Conclusion/Significance Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells

Эффекты новых тяжелых промежуточных бозонов и аномальных калибровочных констант связи и их определение из экспериментов на большом адронном коллайдере

Отчет о НИР (заключительный). Руководитель темы А. А. Панков

Partial purification and identification of protease.

<p>Chromatographic profile of ammonium sulphate precipitated crude proteins from culture supernatants of CHA6.8Δ<i>prtV</i> strain loaded onto an anion exchange column (DE-52). A) Proteins eluted in the non-binding fraction (NB), B) proteins eluted with 0.1 M NaCl, C) proteins eluted with 0.3 M NaCl, +/− shows presence or absence of protease activity, D) azocasein assay with pooled samples (30 µg) NB, 0.1 M#1, 0.1 M #2, 0.3 M and crude proteins. E) Native PAGE profile (lane 1) of crude proteins of CHA6.8Δ<i>prtV</i> strain and (lane 2) of partially purified protease (NB) from DE-52 column. The marked protein band was analyzed by MS/MS sequencing and the peptides highlighted showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. F) The underlined GDSGGP are the amino acid sequences around the serine residue present in trypsin-like serine proteases. G) Protease inhibition test of NB fraction (5 µg) with protease inhibitors 10 mM EDTA, 25 mM PMSF, 25 mM PMSF and 10 mM EDTA, 10 mM EDTA and 20 mM CaCl₂, 10 mM EGTA, 1 µg/ml aprotinin, 28 mM E64, 1 µg/ml leupeptin and 10 mM 1,10-phenanthroline incubated for 30 mins at 37°C. Residual protease activity was assayed by azocasein assay. Twenty-five mM Tris-HCl was used as a negative control. The values shown are the means with standard deviations from three experiments.</p

Histopathological study of ileal tissues.

<p>Panels show photomicrographs of histology of rabbit ileal loop tissue after treatment with A) Partially purified serine protease from <i>V. cholerae</i> strain CHA6.8Δ<i>prtV</i> showing hemorrhagic fluid accumulation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013122#pone-0013122-g004" target="_blank">Fig. 4A</a>, NB). Gross damage of the villus surface structure was observed with hemorrhage in all layers of the mucosa. Magnification, 20X. B) Almost normal villous architecture observed in ileal tissues treated with 50 µg of partially purified protease inhibited with 25 mM PMSF and 10 mM EDTA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013122#pone-0013122-g004" target="_blank">Fig. 4A</a>, NB+PMSF+EDTA). This photomicrograph shows no gross alteration in villus structure but villus lamina propria are slightly dilated and RBC have accumulated at a few places in the basal area. Magnification, 20X. C) Ileal tissues treated with 25 mM Tris-HCl buffer with PMSF and EDTA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013122#pone-0013122-g004" target="_blank">Fig. 4A</a>, control) showed normal villus structure. Magnification 20X. (D) ileal tissues treated with culture supernatant from C6709 strain showed presence of hemorrhage in all layers of the gut mucosa specially in the submucosal layer, Magnification 20X. E) ileal tissues treated with culture supernatant from CHA6.8 strain showed widely dialated villi with rupture at places with gross hemorrhage and inflammatory cells in mucosa and submucosa, Magnification 20X. F) Ileal tissues treated with culture supernatant of CHA6.8Δ<i>prtV</i> strain also showing dilated villi with gross hemorrhage in all layers of the mucosa. Magnification 20X. G) The same section in higher magnification 40X showing ruptured villi with hemorrhage and inflammatory cells in mucosa and submucosa. (H) ileal tissues treated with culture supernatant from CHA6.8Δ<i>prtV</i>ΔVC1649 strain showing villous architecture almost normal with minimum hemorrhage in mucosa and submucosa. (I) TSB treated ileal tissue showing normal gut mucosa.</p

Protease activity assay.

<p>A) Azocasein assay with 30 µg of ammonium sulphate precipitated proteins from culture supernatants of C6709, CHA6.8, CHA6.8Δ<i>prtV</i> and CHA6.8Δ<i>prtV</i>ΔVC<i>1649</i> and inhibition test with 25 mM PMSF, 10 mM EDTA and 10 mM 1,10- phenanthroline. Negative controls were (1) 25 mM Tris-HCl and 25 mM Tris-HCl in the presence of (2) 25 mM PMSF, (3) 10 mM EDTA and (4) 10 mM 1,10- phenanthroline. The values shown are the means with standard deviations from three experiments. B) Skim milk assay for detection of protease in C6709, CHA6.8, CHA6.8Δ<i>prtV</i> and CHA6.8Δ<i>prtV</i>ΔVC<i>1649</i> strains.</p

Rabbit ileal loop assay.

<p>A) RIL response of partially purified protease (50 µg, NB) showing significant hemorrhagic fluid accumulation (FA ratio 1.2+/−0.2 n = 3) and its effect after inhibition with 25 mM PMSF and 10 mM EDTA (NB+PMSF+EDTA) shows significant decrease in fluid accumulation (FA ratio 0.3+/−0.05 n = 3). Twenty five mM Tris-HCl with 25 mM PMSF +10 mM EDTA was used as a negative control (FA ratio = 0.12+/−0.002, n = 3). B) RIL response with culture supernatants of C6709 (FA ratio 1.1+/−0.3, n = 3), CHA6.8 (FA ratio 1.08+/−0.2, n = 3), CHA6.8Δ<i>prtV</i> (FA ratio 1.02+/−0.2, n = 3), CHA6.8Δ<i>prtV</i>ΔVC1649 (FA ratio 0.11+/−0.005, n = 3) and Tryptic soy broth as negative control (FA ratio 0.09+/−0.002, n = 3).</p